Because of the importance of catecholamines in regulating a number of central and peripheral nervous system functions, an understanding of their fundamental neurobiological properties has been of great interest over the past 25 years. One of the intriguing properties of catecholaminergic cells is their capacity to precisely regulate neurotransmitter levels in proportion to the degree to which they are stimulated, thus enabling them to meet secrctory demands. Recent evidence has focused on the phosphorylation and activation of the rate-limiting enzyme in the catecholamine biosynthetic pathway, tyrosine hydroxylase, as the principal mechanism of this regulatory process. Whereas several laboratories have demonstrated that phosphorylation of tyrosine hydroxylase increases the catalytic activity of the enzyme in vitro, our research has focused on studying the significance of this mechanism within intact cells. Isolated bovine adrenal chromaffin cells, which appear to possess the fundamental regulatory properties of catecholaminergic cells, have been used as our cell model. Tyrosine hydroxylase is phosphorylated within the intact chromaffin cell on four serine sites. In response to acetylcholine there is an increased phosphorylation on two of the same serine sites as previously described in vitro (serines 19 and 40) as well as on an additional serine site (serine 31). Importantly, significant disparities are observed between phosphorylation of tyrosine hydroxylase, activation of the enzyme, and stimulation of catecholamine biosynthesis in response to this cholinergic stimulus. The goal of the studies described in this application is to continue this research in an effort to further elucidate the cellular and molecular mechanisms which regulate catecholamine biosynthesis. 1) The identity of the serine on the one remaining tryptic phosphopeptide of tyrosine hydroxylase which has not yet been sequenced will be established. The hypothesis that this phosphopeptidc corresponds to serine 8 and is phosphorylated by the proline directed protein kinase, p34cdc2, will be tested. 2) The identity of the protein kinase responsible for phosphorylating serine 31 will be established; if no known protein kinase is found to be responsible, the serine 31 phosphorylating kinase will be purified from the chromaffin cells and its properties characterized. 3) The stoichiometry of the phosphorylation of each secrine will be determined in resting and stimulated cells. 4) Phosphorylation of tyrosine hydroxylase by purified kinases together with sitedirected mutagenesis of the enzyme, will bc used to establish the influence of each phosphorylation site on catalytic activity. 5) The mechanism, which is responsible for enhancing catecholamine biosynthesis during times when activation of tyrosine hydroxylase is not apparent, will be elucidated. 6) Studies will evaluate the role of calcium/calmodulin-dependent and calcium/ phospholipid-dependent protein kinases in the cholinergic mediated phosphorylation of tyrosine hydroxylase.